Kinetic properties of Rhizopus oryzae RPG1 endo-polygalacturonase hydrolyzing galacturonic acid oligomers

The kinetic characteristics of Rhizopus oryzae endo-polygalacturonase, RPG1, hydrolyzing galacturonic acid oligomers (GalpA)n were determined. RPG1 generates (GalpA)3 as a dominant product of polygalacturonic acid and (GalpA)4–6 hydrolysis. The enzyme can hydrolyze (GalpA)3, but hydrolysis occurs at a significantly lower rate relative to oligomers with a higher degree of polymerization. Hydrolysis of the α-1,4 glycosidic bond by RPG1 is an endothermic process with a ΔHapp, of 1.03±0.04 kcal/mol. Determination of kinetic constants by isothermal titration calorimetry showed that for oligomers (GalpA)3–6, the Km decreased and the kcat increased as the length of the (GalpA) oligomer increased. Fixed time point assays followed by chromatographic analysis provided apparent kcat values similar to those found using isothermal titration calorimetry. Assays to determine to what extent the enzyme is subject to product inhibition demonstrated that the enzyme is competitively inhibited by (GalpA)2 when using (GalpA)4 as substrate. The apparent Ki of 767 μM is significantly higher than the Km values obtained for the series of galacturonic acid oligomers.